Amine-N-oxide redox indicators for fluorimetric determination of analytes

ABSTRACT

A reagent for detecting an analyte by a redox reaction and a fluorimetric determination is disclosed. The reagent comprises a compound of formula (I): 
                         
wherein R 1  and R 2  are each independently selected from R, (CH 2 CH 2 O) m R, COR, COOR, and OCOR; each R 3  is independently selected from NO 2 , CN, R, OR, OCOR, COOR, SR, and halogen; R is H or C 1 -C 4  alkyl, where alkyl is optionally substituted with one or more functional group independently selected from the group consisting of halogen, OR, SR, NR 2 , COOR, CONR 2 , SO 3 R and salts thereof, and PO(OR) 3  and salts thereof; m is an integer from 1 to 20; and n is 1, 2, or 3. The reagent may further comprise an enzyme or a coenzyme for reducing or oxidizing an analyte.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 10/771,872, filed Feb. 4, 2004, now U.S. Pat. No. 7,723,121. Thepresent application claims priority under 35 U.S.C. §119 to GermanApplication No. 103 04448.5 filed Feb. 4, 2003.

BACKGROUND

The invention concerns methods and reagent kits for the fluorimetricdetermination of analytes.

There are numerous methods for determining analytes for example fordiagnostic applications. One method is to determine the analyte by meansof a redox reaction and a redox indicator. In this case an oxidizing orreducing system acts directly on the redox indicator or via a mediator.The presence of the analyte leads to a reduction or oxidation of theredox indicator which enables a qualitative or quantitativedetermination to be carried out.

Depending on the type of redox indicator that is used, the indicator canbe determined by a colorimetric, fluorimetric or electrochemicaldetection method. Examples of colorimetric detection reagents areheteropolyacids (EP-B-0 431 456), tetrazolium compounds (EP-B-0 574769), nitrosoaromatic compounds (EP-A-0 620 283), RIND compounds (EP-B-0190 740), phenazines (WO 93/06487) and indanthrones (EP-B-0 831 327).Examples of electrochemical detection reagents are nitrosoaromatics,phenazines, potassium hexacyanoferrate and benzoquinones (cf. e.g.EP-A-0 441 222 and EP-A-0 505 494). Examples of fluorimetric detectionreagents are e.g. resazurin (U.S. Pat. No. 5,912,139), transition metalcomplexes (Ryabov et al., JBIC 4 (1999) 175-182; Woltman et al., Anal.Chem. 71 (1999) 1504-1512) and scopoletin, esculetin,p-hydroxyphenylacetic acid, di-chlorofluorescein,N-acetyl-3,7-dihydroxyphenoxazine and MNBDH which are used exclusivelyfor the detection of H₂O₂ (see also R. Haughland, Handbook ofFluorescent Probes and Research Chemicals, 6^(th) edition 1996).

However, the fluorimetric detection reagents known from the prior arthave some disadvantages. Thus most known fluorescent indicators requirethat metabolites such as glucose are determined by detecting H₂O₂generated by glucose oxidase. This reaction usually has to becatalytically supported by the enzyme peroxidase and is very prone tointerference by electron donors such as urea or bilirubin. The reagentsare also not stable for long time periods.

In contrast, redox indicators that allow an oxygen-independent detectionof glucose i.e. which directly accept an electron from an oxidizingenzyme instead of oxygen, are advantageous. However, only resazurin andOs and Ru complexes are known to be suitable electron acceptors forthis. However, in the case of resazurin the emission bands of theresorufin formed by the redox reaction strongly overlap the absorptionbands of non-reacted resazurin which considerably reduces thesensitivity of the analyte determination. The high redox potential oftransition metal complexes (e.g. Ru complexes) results in a stronginterference by compounds such as ascorbic acid. Their fluorescenceefficiency also varies with the oxygen content of the sample.

Furthermore in the case of the previously known fluorescent indicatorsthe excitation light sources used are mainly limited to the UV and greenrange of light. Thus for example an inadequate number of compounds areknown which allow use of the particularly strong blue and red LEDs.

SUMMARY

Hence one object of the present invention was to provide newredox-active compounds as detection reagents for the fluorimetricdetermination of analytes which enable the disadvantages of the priorart to be at least partially eliminated.

This object is achieved according to the invention by providing theN-oxide of NBD-amine or derivatives thereof as redox indicators. NBD isthe abbreviated form of “7-nitrobenzo-2-oxa-1,3-diazol.” The NBD-amineformed by reduction is characterized by a high fluorescence and can bevery readily excited with blue light radiation.

Hence a first aspect of the present invention is a method for detectingan analyte by a redox reaction and a fluorimetric determinationcharacterized in that a sample containing the analyte is contacted witha detection reagent which contains a compound of the general formula (I)as a fluorimetric redox indicator:

-   in which R¹ and R² are each independently selected from R,    (CH₂CH₂O)_(m)R, COR, COOR and OCOR,-   R³ in each case is independently selected from NO₂, CN, R, OR, OCOR,    COOR, SR and halogen,-   R is H or C₁-C₄ alkyl, where alkyl is optionally substituted with    halogen, OR, SR, NR₂, COOR, CONR₂, SO₃R and salts thereof or/and    PO(OR)₃ and salts thereof, m is an integer from 1-20, preferably    from 1-10 and n is 1,2 or 3.

Another aspect of the invention is a reagent for detecting an analyte bya redox reaction and a fluorimetric determination which contains acompound of the general formula (I) as described above as thefluorimetric redox indicator.

The present invention is suitable for detecting any analytes that can bedetermined by a redox reaction. The detection can be qualitative,semi-quantitative or quantitative. In one embodiment of the inventionthe analyte can be a reducible or oxidizable substance, for example ametabolite present in a body fluid such as blood, serum, plasma, urineetc. In this case it is expedient to use a detection reagent which, inaddition to the redox indicator, also contains one or more enzymes forreducing or oxidizing the analyte and optionally coenzymes such asnicotine nucleoside derivatives e.g. NAD⁺, NADP⁺ or flavin nucleosidederivatives e.g. FAD. Preferred examples of such analytes are glucose,lactate, alcohol, galactose, cholesterol, fructose, glycerol, pyruvate,creatinine, alanine, phenylalanine, leucine, triglycerides,HDL-cholesterol. Glucose can for example be detected by known methodsusing glucose oxidase (GOD), glucose dye oxidoreductase (GlucDOR) orglucose dehydrogenase (GDH)/diaphorase.

Furthermore the analyte may also be an enzyme that catalyses a redoxreaction, for example an oxidoreductase such as glucose oxidase, glucosedye oxidoreductase, dehydrogenases or an enzyme whose reaction can becoupled to an oxidoreductase reaction.

In addition to the redox indicator and, if required, an enzyme forreducing or oxidizing the analyte, the detection reagent canadditionally contain common components such as coenzymes, auxiliarysubstances, buffers and optionally mediators. Substances are suitable asmediators which support the acceptance of electrons by the redoxindicator (I). However, in general those redox indicators are preferredwhich can directly accept electrons.

The method according to the invention is carried out in conventionaltest formats such as in dry or wet tests. In a dry test an absorbentmaterial, e.g. in the form of a test strip, is used as a support onwhich the detection reagent can be applied in a dry form e.g. as alyophilisate. Liquid tests are carried out in a liquid phase in suitablereaction vessels e.g. cuvettes, microtitre plates etc. where thedetection reagent can be provided in the reaction vessel itself or inseparate containers in a dry or liquid form.

For a fluorimetric determination, the sample is irradiated withexcitation light of a predetermined wavelength and the fluorescenceemission light emitted by the sample that has a different wavelength isdetermined by known methods. Suitable variation of the substituents R¹ ,R² and R³ enables the present invention to provide optimized testformats for the determination of any analytes.

It is preferable that the redox indicator (I) has one or morehydrophilic groups, e.g. OR groups, COOR groups etc., to increasesolubility. In a particularly preferred embodiment R¹ and R² are C₁₋₂alkyl groups substituted with OR such as hydroxyethyl groups orpolyoxyethylene groups. R³ is preferably NO₂ and n is 1. A particularlypreferred example of a redox indicator according to the invention isshown in FIG. 1.

Another aspect of the invention is a reagent for detecting an analyte bya redox reaction and a fluorimetric determination comprising a compoundof the general formula (I) as stated above as the redox indicator.

In addition to the redox indicator, the reagent according to theinvention can also contain other components selected from enzymes,coenzymes, auxiliary substances, buffers and mediators.

The present invention is further elucidated by the following figures andthe example.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the N-oxide of the NBD-amine as an example of a redoxindicator according to the invention.

FIG. 2 shows the kinetics of the NBD-amine-N-oxide reduction in a systemfor detecting glucose at various glucose concentrations.

DETAILED DESCRIPTION EXAMPLE Glucose determination using anNBD-amine-N-oxide as the redox indicator.

The following compounds were added to a 3 ml fluorescence cuvette (thestated concentrations refer to the final concentration in the cuvette;the N-oxide of the NBD-amine was prepared according to P. B. Ghosh, M.W. Whitehouse, J. Med. Chem., 11, 305-311 (1968)):

glucose dehydrogenase (GlucDH): 1.3 U/ml diaphorase: 1.3 U/ml NAD⁺: 36.9μmol/l N-oxide of the NBD-amine: 35.4 μmol/l

The reaction was started by adding an aqueous glucose solution (0.1 Mphosphate buffer, pH 7.4 containing 1% NaCl). The kinetics of thereaction were recorded for various glucose concentrations at anexcitation wavelength of 470 nm and an emission wavelength of 560 nm.The result of the experiment is shown in FIG. 2.

FIG. 2, in which the intensity of the fluorescence signal (intensity) inimpulses per second (cps) is plotted versus time in seconds (sec), showsthat an increase in fluorescence is found which is proportional to theglucose concentration present in the sample. In this case the measuringcurves 1 to 5 correspond to glucose concentrations of 0, 0.06, 1.2, 2.4and 4 (each in mg/dl).

It is noted that terms like “preferably”, “commonly”, and “typically”are not utilized herein to limit the scope of the claimed invention orto imply that certain features are critical, essential, or evenimportant to the structure or function of the claimed invention. Rather,these terms are merely intended to highlight alternative or additionalfeatures that may or may not be utilized in a particular embodiment ofthe present invention.

For the purposes of describing and defining the present invention it isnoted that the term “substantially” is utilized herein to represent theinherent degree of uncertainty that may be attributed to anyquantitative comparison, value, measurement, or other representation.The term “substantially” is also utilized herein to represent the degreeby which a quantitative representation may vary from a stated referencewithout resulting in a change in the basic function of the subjectmatter at issue.

Having described the invention in detail and by reference to specificembodiments thereof, it will be apparent that modifications andvariations are possible without departing from the scope of theinvention defined in the appended claims. More specifically, althoughsome aspects of the present invention are identified herein as preferredor particularly advantageous, it is contemplated that the presentinvention is not necessarily limited to these preferred aspects of theinvention.

What is claimed is:
 1. A reagent kit that detects a reducible oroxidizable analyte by a redox reaction and a fluorimetric determination,the reagent kit comprising: (a) one or more enzymes that reduce oroxidize the reducible or oxidizable analyte; and (b) a redox indicatorthat is oxidized or reduced in the presence of both (i) the reducible oroxidizable analyte, and (ii) the one or more enzymes by accepting anelectron, directly or via a mediator, from the reducible or oxidizableanalyte, the redox indicator being a compound of formula (I):

wherein R¹ and R² are each independently selected from R,(CH₂CH₂O)_(m)R, COR, COOR, and OCOR; each R³ is independently selectedfrom NO₂, CN, R, OR, OCOR, COOR, SR, and halogen; R is H or C₁-C₄ alkyl,where alkyl is optionally substituted with one or more functional groupindependently selected from the group consisting of halogen, OR, SR,NR₂, COOR, CONR₂, SO₃R and salts thereof, and PO(OR)₃ and salts thereof;m is an integer from 1 to 20; and n is 1, 2, or 3 wherein the reducibleor oxidzable analyte is glucose and the one or more enzymes is selectedfrom the group consisting of glucose oxidase, glucose dyeoxidoreductase, and glucose dehydrogenase/diaphorase.
 2. The reagent kitof claim 1, further comprising components selected from coenzymes,auxiliary substances, buffers, and mediators.
 3. The reagent kit ofclaim 1, wherein R¹ and R² are a C₁-C₂ alkyl group substituted with OH.4. The reagent kit of claim 1, wherein R³ is NO₂.
 5. The reagent kit ofclaim 4, wherein n is
 1. 6. The reagent kit of claim 1, wherein thecompound of formula (I) is


7. The reagent kit of claim 1, wherein the redox indicator is oxidizedor reduced by directly accepting an electron from the reducible oroxidizable analyte.
 8. A reagent kit that detects an analyte by a redoxreaction and a fluorimetric determination, the reagent kit comprising:(a) one or more enzymes that reduce or oxidize the reducible oroxidizable analyte; and (b) a redox indicator that is oxidized orreduced in the presence of (i) the reducible or oxidizable analyte, and(ii) the one or more enzymes, the redox indicator being a compound offormula (I):

where R¹ and R² are each independently selected from R, (CH₂CH₂O)_(m)R,COR, COOR, and OCOR; each R³ is independently selected from NO₂, CN, R,OR, OCOR, COOR, SR, and halogen; R is H or C₁-C₄ alkyl, where alkyl isoptionally substituted with one or more functional group independentlyselected from the group consisting of halogen, OR, SR, NR₂, COOR, CONR₂,SO₃R and salts thereof, and PO(OR)₃ and salts thereof; m is an integerfrom 1 to 20; and n is 1, 2, or 3, wherein, the reducible or oxidizableanalyte is selected from the group consisting of glucose, lactate,alcohol, galactose, cholesterol, fructose, glycerol, pyruvate,creatinine, alanine, phenyalanine, leucine, triglycerides,HDL-cholesterol, glutamate-oxalacetate transaminase, glutamate-pyruvatetransaminase, alanine aminotransferase, lactate dehydrogenase, andcreatine kinase wherein the reducible or oxidzable analyte is glucoseand the one or more enzymes is selected from the group consisting ofglucose oxidase, glucose dye oxidoreductase, and glucosedehydrogenase/diaphorase.
 9. The reagent kit of claim 8, wherein theredox indicator is oxidized or reduced by directly accepting an electronfrom the reducible or oxidizable analyte.
 10. The reagent kit of claim8, wherein the redox indicator is oxidized or reduced by accepting anelectron, directly or via a mediator, from the reducible or oxidizableanalyte.
 11. The reagent kit of claim 8, wherein the redox indicator isoxidized or reduced by directly accepting an electron from the reducibleor oxidizable analyte.
 12. The reagent kit of claim 8, wherein R¹ and R²are a C₁-C₂ alkyl group substituted with OH.
 13. The reagent kit ofclaim 8, wherein R³ is NO₂.
 14. The reagent kit of claim 13, wherein nis
 1. 15. The reagent kit of claim 8, wherein the compound of formula(I) is